The ionophore X-537A has been shown to induce profound physiological changes in cardiac activity characteristic of positive inotropism, but the biochemical basis of these effects remain poorly understood. Characterization of the complexes formed between X-537A and mono- and divalent cations, and catecholamines in membranous phospholipid bilayer vesicles should provide information essential for design of a therapeutic application in cases of cardiac insufficiency. We hope to characterize the thermodynamic properties of inorganic cation complexes of the ionophore in the vesicle system, employing techniques which we have already developed for bulk organic solvents of various polarities. We will use fluorescence and circular dichroism as measures of complex formation to determine stoichiometries and equilibrium constants in vesicles. Kinetic studies will be continued in bulk solvents, employing nuclear magnetic resonance to calculate exchange rates, and stopped-flow fluorescence spectroscopy to monitor binding and substitution reactions. We also plan to use fluorescence correlation spectroscopy to obtain kinetic data for translational diffusion and for complex formation and dissociation rate constants in vesicles. The rotational motion of X-537A in membranes will be studied by fluorescence depolarization, making use of the marked temperature dependence of fluidity in bilayers of different homogeneous phospholipid compositions. Such temperature effects will be correlated with electron spin resonance measurements of spin labeled lipid probes which may be simultaneously introduced into the bilayer. We intend to pursue the initial observations of fusogenic properties of X-537A by observing its effects on membrane structure. Electron microscopy, employing primarily negative staining procedures, will provide a direct measure of an increase in vesicle volume induced by X-537A. Effects of the ionophore on membrane fluidity may be observed with ESR, and kinetics of membrane fusion may be followed by light scattering.